hybpiper-nf and paragone-nf: Containerization and additional options for target capture assembly and paralog resolution.

Premise: The HybPiper pipeline has become one of the most widely used tools for the assembly of target capture data for phylogenomic analysis. After the production of locus sequences and before phylogenetic analysis, the identification of paralogs is a critical step for ensuring the accurate inference of evolutionary relationships. Algorithmic approaches using gene tree topologies for the inference of ortholog groups are computationally efficient and broadly applicable to non-model organisms, especially in the absence of a known species tree. Methods and Results: We containerized and expanded the functionality of both HybPiper and a pipeline for the inference of ortholog groups, providing novel options for the treatment of target capture sequence data, and allowing seamless use of the outputs of the former as inputs for the latter. The Singularity container presented here includes all dependencies, and the corresponding pipelines (hybpiper-nf and paragone-nf, respectively) are implemented via two Nextflow scripts for easier deployment and to vastly reduce the number of commands required for their use. Conclusions: The hybpiper-nf and paragone-nf pipelines are easily installed and provide a user-friendly experience and robust results to the phylogenetic community. They are used by the Australian Angiosperm Tree of Life project. The pipelines are available at https://github.com/chrisjackson-pellicle/hybpiper-nf and https://github.com/chrisjackson-pellicle/paragone-nf.

Pollen DNA metabarcoding reveals cryptic diversity and high spatial turnover in alpine plant-pollinator networks.

Alpine plant-pollinator communities play an important role in the functioning of alpine ecosystems, which are highly threatened by climate change. However, we still have a poor understanding of how environmental factors and spatiotemporal variability shape these communities. Here, we investigate what drives structure and beta diversity in a plant-pollinator metacommunity from the Australian alpine region using two approaches: pollen DNA metabarcoding (MB) and observations. Individual pollinators often carry pollen from multiple plant species, and therefore we expected MB to reveal a more diverse and complex network structure. We used two gene regions (ITS2 and trnL) to identify plant species present in the pollen loads of 154 insect pollinator specimens from three alpine habitats and construct MB networks, and compared them to networks based on observations alone. We compared species and interaction turnover across space for both types of networks, and evaluated their differences for plant phylogenetic diversity and beta diversity. We found significant structural differences between the two types of networks; notably, MB networks were much less specialized but more diverse than observation networks, with MB detecting many cryptic plant species. Both approaches revealed that alpine pollination networks are very generalized, but we estimated a high spatial turnover of plant species (0.79) and interaction rewiring (0.6) as well as high plant phylogenetic diversity (0.68) driven by habitat differences based on the larger diversity of plant species and species interactions detected with MB. Overall, our findings show that habitat and microclimatic heterogeneity drives diversity and fine-scale spatial turnover of alpine plant-pollinator networks.

New targets acquired: Improving locus recovery from the Angiosperms353 probe set.

PREMISE: Universal target enrichment kits maximize utility across wide evolutionary breadth while minimizing the number of baits required to create a cost-efficient kit. The Angiosperms353 kit has been successfully used to capture loci throughout the angiosperms, but the default target reference file includes sequence information from only 6-18 taxa per locus. Consequently, reads sequenced from on-target DNA molecules may fail to map to references, resulting in fewer on-target reads for assembly, and reducing locus recovery. METHODS: We expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a 'mega353' target file, with each locus represented by 17-373 taxa. This mega353 file is a drop-in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user-selected taxon groups, and to incorporate other transcriptome or protein-coding gene data sets. RESULTS: Compared to the default Angiosperms353 file, the mega353 file increased the percentage of on-target reads by an average of 32%, increased locus recovery at 75% length by 49%, and increased the total length of the concatenated loci by 29%. DISCUSSION: Increasing the phylogenetic density of the target reference file results in improved recovery of target capture loci. The mega353 file and associated scripts are available at: https://github.com/chrisjackson-pellicle/NewTargets.

Implications of the 2019-2020 megafires for the biogeography and conservation of Australian vegetation.

Australia's 2019-2020 'Black Summer' bushfires burnt more than 8 million hectares of vegetation across the south-east of the continent, an event unprecedented in the last 200 years. Here we report the impacts of these fires on vascular plant species and communities. Using a map of the fires generated from remotely sensed hotspot data we show that, across 11 Australian bioregions, 17 major native vegetation groups were severely burnt, and up to 67-83% of globally significant rainforests and eucalypt forests and woodlands. Based on geocoded species occurrence data we estimate that >50% of known populations or ranges of 816 native vascular plant species were burnt during the fires, including more than 100 species with geographic ranges more than 500 km across. Habitat and fire response data show that most affected species are resilient to fire. However, the massive biogeographic, demographic and taxonomic breadth of impacts of the 2019-2020 fires may leave some ecosystems, particularly relictual Gondwanan rainforests, susceptible to regeneration failure and landscape-scale decline.

Genetic rescue in a plant polyploid complex: Case study on the importance of genetic and trait data for conservation management.

Knowledge of the biology of rare plant species is indispensable to aid their survival and to inform efficient conservation actions, but in many cases relevant data are lacking. In addition, while studies of conservation genetics have provided a wealth of information on the considerations arising from inbreeding, mate limitation, or local adaptation, the impact of intraspecific polyploidy remains understudied. In this study, we examined the breeding system of the rare Australian daisy Rutidosis lanata (Asteraceae) and screened ten of its populations for their ploidy level to develop recommendations for management actions, in particular, with regard to seed sourcing and genetic rescue. We found R. lanata to represent a polyploid complex, with tetraploid, pentaploid and hexaploid individuals coexisting in the same species. Crossing experiments confirmed R. lanata to be self-incompatible. Mate availability varied from c. 49% to c. 76% across populations. Most populations showed mate availability of c. 50%-70%, suggesting that mate limitation resulting from a lack of local genetic diversity may cause or at least contribute to reduced seed set. Crossing between populations resulted in significantly higher reproductive success for all populations except one, suggesting the possibility of genetic rescue through population mixing. However, the crossing experiments also showed that pentaploids suffer from a severely reduced paternal reproductive fitness. Any additional hybrids between tetraploids and pentaploids, as would be created by mixing populations with different genome copy numbers during conservation work, would consequently exacerbate mate limitation and thus reduce population viability. We conclude that seed set and thus population viability can be maximized by mixing populations with the same number of genome copies, but that populations with different numbers should be kept spatially separated. The case of Rutidosis lanata provides an example and a potential template for examining the conservation genetics of other species that may constitute polyploid complexes.

Species trees from consensus single nucleotide polymorphism (SNP) data: Testing phylogenetic approaches with simulated and empirical data.

Datasets of hundreds or thousands of SNPs (Single Nucleotide Polymorphisms) from multiple individuals per species are increasingly used to study population structure, species delimitation and shallow phylogenetics. The principal software tool to infer species or population trees from SNP data is currently the BEAST template SNAPP which uses a Bayesian coalescent analysis. However, it is computationally extremely demanding and tolerates only small amounts of missing data. We used simulated and empirical SNPs from plants (Australian Craspedia, Asteraceae, and Pelargonium, Geraniaceae) to compare species trees produced (1) by SNAPP, (2) using SVD quartets, and (3) using Bayesian and parsimony analysis with several different approaches to summarising data from multiple samples into one set of traits per species. Our aims were to explore the impact of tree topology and missing data on the results, and to test which data summarising and analyses approaches would best approximate the results obtained from SNAPP for empirical data. SVD quartets retrieved the correct topology from simulated data, as did SNAPP except in the case of a very unbalanced phylogeny. Both methods failed to retrieve the correct topology when large amounts of data were missing. Bayesian analysis of species level summary data scoring the two alleles of each SNP as independent characters and parsimony analysis of data scoring each SNP as one character produced trees with branch length distributions closest to the true trees on which SNPs were simulated. For empirical data, Bayesian inference and Dollo parsimony analysis of data scored allele-wise produced phylogenies most congruent with the results of SNAPP. In the case of study groups divergent enough for missing data to be phylogenetically informative (because of additional mutations preventing amplification of genomic fragments or bioinformatic establishment of homology), scoring of SNP data as a presence/absence matrix irrespective of allele content might be an additional option. As this depends on sampling across species being reasonably even and a random distribution of non-informative instances of missing data, however, further exploration of this approach is needed. Properly chosen data summary approaches to inferring species trees from SNP data may represent a potential alternative to currently available individual-level coalescent analyses especially for quick data exploration and when dealing with computationally demanding or patchy datasets.

Both morph- and species-dependent asymmetries affect reproductive barriers between heterostylous species.

The interaction between floral traits and reproductive isolation is crucial to explaining the extraordinary diversity of angiosperms. Heterostyly, a complex floral polymorphism that optimizes outcrossing, evolved repeatedly and has been shown to accelerate diversification in primroses, yet its potential influence on isolating mechanisms remains unexplored. Furthermore, the relative contribution of pre- versus postmating barriers to reproductive isolation is still debated. No experimental study has yet evaluated the possible effects of heterostyly on pre- and postmating reproductive mechanisms. We quantify multiple reproductive barriers between the heterostylous Primula elatior (oxlip) and P. vulgaris (primrose), which readily hybridize when co-occurring, and test whether traits of heterostyly contribute to reproductive barriers in unique ways. We find that premating isolation is key for both species, while postmating isolation is considerable only for P. vulgaris; ecogeographic isolation is crucial for both species, while phenological, seed developmental, and hybrid sterility barriers are also important in P. vulgaris, implicating sympatrically higher gene flow into P. elatior. We document for the first time that, in addition to the aforementioned species-dependent asymmetries, morph-dependent asymmetries affect reproductive barriers between heterostylous species. Indeed, the interspecific decrease of reciprocity between high sexual organs of complementary floral morphs limits interspecific pollen transfer from anthers of short-styled flowers to stigmas of long-styled flowers, while higher reciprocity between low sexual organs favors introgression over isolation from anthers of long-styled flowers to stigmas of short-styled flowers. Finally, intramorph incompatibility persists across species boundaries, but is weakened in long-styled flowers of P. elatior, opening a possible backdoor to gene flow through intramorph pollen transfer between species. Therefore, patterns of gene flow across species boundaries are likely affected by floral morph composition of adjacent populations. To summarize, our study highlights the general importance of premating isolation and newly illustrates that both morph- and species-dependent asymmetries shape boundaries between heterostylous species.

Quantifying phytogeographical regions of Australia using geospatial turnover in species composition.

The largest digitized dataset of land plant distributions in Australia assembled to date (750,741 georeferenced herbarium records; 6,043 species) was used to partition the Australian continent into phytogeographical regions. We used a set of six widely distributed vascular plant groups and three non-vascular plant groups which together occur in a variety of landscapes/habitats across Australia. Phytogeographical regions were identified using quantitative analyses of species turnover, the rate of change in species composition between sites, calculated as Simpson's beta. We propose six major phytogeographical regions for Australia: Northern, Northern Desert, Eremaean, Eastern Queensland, Euronotian and South-Western. Our new phytogeographical regions show a spatial agreement of 65% with respect to previously defined phytogeographical regions of Australia. We also confirm that these new regions are in general agreement with the biomes of Australia and other contemporary biogeographical classifications. To assess the meaningfulness of the proposed phytogeographical regions, we evaluated how they relate to broad scale environmental gradients. Physiographic factors such as geology do not have a strong correspondence with our proposed regions. Instead, we identified climate as the main environmental driver. The use of an unprecedentedly large dataset of multiple plant groups, coupled with an explicit quantitative analysis, makes this study novel and allows an improved historical bioregionalization scheme for Australian plants. Our analyses show that: (1) there is considerable overlap between our results and older biogeographic classifications; (2) phytogeographical regions based on species turnover can be a powerful tool to further partition the landscape into meaningful units; (3) further studies using phylogenetic turnover metrics are needed to test the taxonomic areas.

Phylogenetic relationships of the Australasian shrubby everlastings Ozothamnus and Cassinia (Asteraceae: Asteroideae: Gnaphalieae).

The first comprehensive phylogenetic study of the Australasian shrubby everlastings Ozothamnus, Cassinia, and their satellite genera is presented based on the nuclear ribosomal external and internal transcribed spacer and three chloroplast spacer regions (matK-psbA, psbA-trnH, ycf6-pbsM). While the hypothesis of the monophyly of the sequence copies found in Cassinia cannot be rejected, sequences from Ozothamnus are found to be non-monophyletic in three possible taxonomic circumscriptions of that genus. Cassinia, Calomeria, Odixia and Haeckeria are nested in a narrowly circumscribed Ozothamnus. Ozothamnus section Hebelaena, a sub-shrubby group long recognized as potentially misplaced in the genus, is more heterogeneous than expected and is found to be potentially polyphyletic. Perhaps more surprisingly, a group of four species previously considered part of "core" Ozothamnus is found to be more closely related to other genera. It includes the subalpine "cascade everlastings" of south-eastern Australia. Although many characters traditionally used for the delimitation of genera are homoplasious, connivent involucral bracts and presence of paleae are synapomorphies for Cassinia. Except for the robust shrubby habit, no morphological synapomorphy for core Ozothamnus/Cassinia is currently evident. Future studies are needed to test these results with additional data, to identify morphological and anatomical synapomorphies for individual clades and to resolve species-level relationships with more extensive sampling to address the possibilities of incomplete lineage sorting or reticulate evolution.

Phylogenetic analysis of Primula section Primula reveals rampant non-monophyly among morphologically distinct species.

The type section of Primula (Primulaceae), here considered to include seven species, is phylogenetically quite isolated in its genus. Although its species are popular ornamentals, traditional medicinal plants and model organisms for the study of heterostyly, the section has not yet been studied from a phylogenetic or evolutionary perspective. Using phylogenetic analysis of nuclear ITS and plastid data from all species and subspecies, we find that widespread Primula elatior is genetically heterogeneous and non-monophyletic to most if not all of the other ingroup taxa. The Genealogical Sorting Index (GSI) indicates that the assumption of all currently accepted species being independent lineages is consistent with the data. It is possible that P. elatior in its current circumscription may represents the disjointed remnant of an ancestral species from which the other recognized species diverged. However, based on available data, the alternative possibility of introgression explaining the non-monophyly of this species cannot be excluded. Species trees show P. elatior and P. veris as sister species. Primula vulgaris and P. juliae are closely related, while, in contrast to previous assumptions, P. renifolia does not appear to be a close relative of P. megaseifolia. With the section's isolation from the rest of the genus and very short internal branches, our dataset also presents a case study of the confounding effects of different branch length priors on the Bayesian estimation of resulting branch length estimates. Experimental runs using different priors confirm the problem of resulting estimates varying by orders of magnitude, while topology and relative branch lengths seem unaffected.

Fallacies and false premises-a critical assessment of the arguments for the recognition of paraphyletic taxa in botany.

One of the central controversies in contemporary taxonomy and systematics revolves around whether to accept or to reject paraphyletic taxa. The present review is the result of a survey of the ongoing discussion in botany over the past ca. 15 years, and attempts to systematically and critically assess all individual arguments presented for the formal recognition of paraphyletic groups in the classification of life. Where arguments are found to be without merit, rebuttals are presented in the hope of excluding them from further discussion, which can then concentrate on those that have merit. Where arguments are found to be sound, their implications and possible solutions are discussed. The controversy around paraphyletic taxa can be broken down into three questions: whether their rejection or acceptance would lead to a classification better reflecting patterns of biological diversity and evolutionary history; whether their rejection or acceptance would lead to a more practical, useful and predictive classification; and whether their rejection is compatible with ranked and binary Linnaean taxonomy. All available arguments for paraphyletic taxa relating to the first question are demonstrated to be based on various logical fallacies or false premises, especially misunderstandings of the principles of phylogenetic systematics. The issue of usefulness is harder to resolve, as different classifications serve different needs. It is presumably unavoidable but also preferable that phylogenetic and non-phylogenetic ways of classifying species continue to coexist, serving different needs. Finally, an insistence on monophyletic taxa is found to be incompatible with binary taxonomy under a set of very specific circumstances and assumptions whose presence and accuracy are not universally accepted. © The Willi Hennig Society 2011.